For decades, it has been recognized that transcribing along the DNA double helix by a RNA polymerase can enhance localized DNA supercoiling. This process has been elegantly explained by a twin-supercoiled- domain model in which positive DNA supercoils are generated ahead of a translocating RNA polymerase and negative supercoils behind it. However, detailed mechanisms at the molecular level are still unavailable. For instance, how DNA topological barriers affect transcription-coupled DNA supercoiling (TCDS) has not been fully understood. The long-term goal of the proposed research is to understand the molecular mechanisms and biological functions of TCDS and DNA topological barriers. The objectives of this application are to determine what properties make certain DNA-looping and -wrapping proteins function as topological barriers and block supercoil diffusion, to study how DNA topological barriers regulate TCDS in E. coli cells, and to examine how localized, transient TCDS activates gene expression in vivo. Our central hypothesis is that TCDS by RNA polymerases is a major chromosome remodeling force in E. coli cells where the cells use nucleoprotein-based, topological barriers to confine TCDS to localized regions and, as a result, greatly influence the nearby, coupled DNA transactions. Our hypothesis has been formulated on the basis of strong preliminary data produced in our laboratory and will be tested by pursuing the following three specific aims: 1) to determine molecular mechanisms of DNA topological barriers and examine how the DNA topological barriers confine supercoils to localized regions and affect the efficiency of TCDS; 2) to examine the role of DNA topological barriers in TCDS in E. coli cells; 3) to investigate how transient, dynamic TCDS activates transcription in E. coli cells and how DNA topological barriers affect the efficiency of transcription activation by TCDS. Information from the proposed experiments will ultimately provide us with a better understanding of the mechanisms of TCDS, especially the roles of DNA topological barriers in TCDS and the coupled gene transcription and expression.